SUMMARY Trophoblast lineage formation represents the first differentiation event in the life cycle of placental mammals. This process begins in the preimplantation embryo and continues in the postimplantation conceptus. Failure to accurately establish the trophoblast lineage has major consequences, including developmental arrest prior to the blastocyst stage, implantation failure, early miscarriage, and placental abnormalities. The overall objective of the proposed studies is to use mouse embryos, and mouse and human embryonic stem (ES) cells as model systems to elucidate the transcriptional mechanisms that govern trophoblast lineage development. Preliminary studies discovered a novel and critical role for transcription factor AP-2? (TFAP2C) in triggering and regulating key events that underlie trophoblast lineage development. The overall hypothesis is that TFAP2C is a key orchestrator of trophoblast lineage formation in mice and humans, whose precise regulation during the window of preimplantation development is critical for postimplantation placental development. Studies in Specific Aim 1 will determine the role of TFAP2C in mouse trophoblast lineage formation. Experiments will test the hypothesis that TFAP2C induces a trophoblast cell-fate in preimplantation embryos by positively regulating the formation of polar-outside cells. Work in Specific Aim 2 will define the molecular mechanism by which TFAP2C induces a trophoblast gene expression program in mice. Experiments will test the hypothesis that TFAP2C establishes a trophoblast gene expression program by forming a regulatory complex with transcription factor TEAD4 and the HIPPO signaling protein YAP1. In Specific Aim 3 the importance of the TFAP2C-mediated mechanisms in mouse preimplantation embryos on subsequent postimplantation placental development will be determined. Experiments will test the hypothesis that precise regulation of Tfap2c during early development is essential for proper continuation of trophoblast progenitor development in postimplantation embryos. Lastly, Specific Aim 4 will elucidate the role of TFAP2C in human trophoblast lineage development. Proposed experiments will test the hypothesis that TFAP2C is required for trophoblast lineage formation and differentiation into functional syncytiotrophoblasts and extravillous trophoblasts. Taken together, results of the proposed studies will provide new insights into the regulatory role of TFAP2C in mouse and human trophoblast lineage development. The knowledge gained from these studies will be highly relevant to understanding the molecular basis of early embryonic loss and clinical reproductive disorders that are associated with abnormal placentation.